Lipids mediate supramolecular outer membrane protein assembly in bacteria

β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

) only 11/14 are shown, with mid-barrel mutants G211, A251, and I295 excluded for clarity with residue labels. Inactive mutants located closer to the trimer interface are also not shown. B, Sites for BPA incorporation in BtuB (PDB ID 1NQF) (86), represented as sticks, are predominantly located around the middle of the -barrel. The 90 o rotation highlights the location of the sites, which sample approximately every third strand around the entirety of the barrel. C, Sites for BPA incorporation in FepA (PDB ID 1FEP) (73), represented as sticks, are predominantly located mid-barrel, with a single mutant (D553 BPA ) located on the extracellular end of a -strand. The 90 o rotation highlights the location of the sites, which sample the entirety of the barrel. Only variants that were purified and characterized for each OMP are displayed. (50 mL) were grown to an OD600 of 0.5 in LB and exposed to UV light (365) on ice. 1 mL of sample was removed at each time point, serially diluted, and plated onto LB/agar plates and incubated at 37 o C overnight. The resulting colonies were counted and logCFUs plotted against time (n=3 biological repeats). B, A colicin-based cytotoxic assay established if BtuB BPA variants were folded and correctly inserted into the OM of E. coli RK5016 cells. ColE9 (0.5-10 µM) was spotted onto plates containing E. coli RK5016 cells transformed with pEVOL-pBpF and individual BtuB BPA variants expressed from a pBAD vector. The lowest ColE9 concentration where cytotoxicity is observed was plotted for each BPA incorporation site. Biological replicates are displayed as  and . Representative images of colicin cytotoxicity assays are shown for three mutants G242 BPA , V523 BPA and W371 BPA . C, Representative transillumination and corresponding epifluorescence images (average of 100 frames, 100 ms exposure) of E. coli RK5016 cells, expressing BtuB BPA variants and labelled with ColE9 AF647 . Images show a punctate labelling pattern consistent with the presence of islands. Exposure of cells to UV for 90 min did not appear to alter the distribution of BtuB relative to wild-type cells and no-UV controls. In the absence of BPA, no fluorescence was observed consistent with the lack of BtuB incorporation in the OM. All images are to the same scale. Scale bar, 2 m. Images are not shown at the same contrast level. were grown to OD600 of 0.5 and exposed to UV light (365) on ice. 1 mL sample was removed at each time point, serially diluted, and plated onto LB/agar plates and incubated at 37 o C overnight.
The resulting colonies were counted and logCFUs plotted against time (n=3 biological repeats).
B, A colicin-based cytotoxic assay was used to determine if FepA BPA variants were functional. ColB (0.5-32 µM) was spotted onto plates with E. coli BW25113 expressing a specific FepA BPA variant (pBad) and tRNase for BPA incorporation (pEVOL-pBpF plasmid). The lowest ColB concentration where cell killing was observed was plotted for each BPA mutant. Biological replicates are displayed as  and . Representative images of colicin cytotoxicity plates are shown for three mutants I255 BPA , L588 BPA and V679 BPA . C, Transillumination and epifluorescence images (average of 100 frames, 100 ms exposure) of E. coli BW25113 cells transformed with the pEVol-pBpF plasmid and pBAD expression plasmid, encoding individual FepA BPA mutants, were grown to a OD600 of 0.6 and protein expression induced with arabinose for 2 h prior to labelling with ColB-GFP. Images show a punctate labelling pattern consistent with the presence of islands.
Exposure of cells to UV did not alter the distribution of FepA, when compared to wild-type and the no UV control. In the absence of BPA, no fluorescence was observed, consistent with the lack of FepA in the OM. Scale bar 1 m. Images are not shown at the same contrast level. assessed by CFU counts. Cells (50 mL) were grown to OD600 0.5 and exposed to UV light (365) on ice. Samples (1 mL) were removed at 30 min intervals, serially diluted, and plated onto LB/agar plates that were incubated at 37 o C overnight. Resulting colonies were counted and logCFUs plotted against time (n=3 biological repeats). B, A colicin-based cytotoxic assay was used to determine if OmpF BPA variants were correctly folded and inserted into the OM of E. coli BZB1107 cells transformed with plasmids encoding tRNase for BPA incorporation (pEVOL-pBpF) and OmpF TAG mutant expressed from a pBAD vector. ColN (0.1-8 µM) was spotted onto plates expressing specific OmpF BPA variants. The lowest ColN concentrations where growth clearance was observed are plotted for each BPA incorporation site. Biological replicates are displayed as  and , for mutants that are resistant to ColN killing such as A321 BPA and V333 BPA , which are located close to the trimer interface, no symbols are shown. Representative images of killing are shown for three mutants V177 BPA , Q277 BPA and A321 BPA . C, Transillumination and epifluorescence images (average of 100 frames, 100 ms exposure) of E. coli BZB1107 cells, transformed with pEVOL-pBpF and pBad plasmids were grown to a OD600 of 0.5 and ompF expression induced with arabinose for 2 h prior to labelling with ColN-mCherry. Images show OmpF is widely distributed around the periphery of the cells. Exposure of cells to UV for 90 min did not alter the distribution of OmpF appreciably. In the absence of BPA, no fluorescence was observed, consistent with the lack of protein in the membrane. Mutations A321 BPA and V333 BPA are located close to the trimer interface, suggesting resistance to ColN killing is due to impaired OmpF insertion into the OM. All images are to the same scale. Scale bar, 2 m. Images are not shown at the same contrast level.    Supplementary Table S2. Complex of phospholipid and the FepA V679 BPA mutant was also observed (panel D). LPS was not detected in these spectra, presumably due to their relatively low abundance. Masses are listed in Supplementary Table S3. Panels are native-MS spectra for OmpF BPA mutants from peak 1 and 2, following UV-activation, membrane extraction, and purification. All samples were injected in a buffer containing 1% -OG and 200 mM ammonium acetate (pH 7.0). Peak 1 samples are to the left, peak 2 samples to the right. A, OmpF V177 BPA /G196 BPA double mutant, B, OmpF G198 BPA , C, OmpF V196 BPA , D, OmpF T238 BPA , E, OmpF L281 BPA . PL-bound OmpF was observed in all peak 1 samples whilst LPS-and PL-bound forms of OmpF were observed in the peak 2 samples. Insets show SDSpolyacrylamide gels of each sample analysed that has been stained with Coomassie blue for protein and ProQ emerald green for LPS. In all cases, only peak 2 samples stain for LPS. Together these data show that BPA localized at central locations within the OmpF -barrel are similarly able to crosslink to PLs or LPS. Spectra for the peak 2 fractions suggest that OmpF crosslinking to LPS facilitates its interactions with the TBDTs FhuA and FepA, which are evident in the native-MS spectra for every mutant. For the V177 BPA /G196 BPA double mutant, we detect up to 6 PLs, and up to two LPS molecules bound to OmpF trimer. Masses are presented in Supplementary Table S4. of OmpF, were observed in both peak 1 and peak 2 samples. For the peak 2 fraction of the OmpF Y202 BPA mutant, charge states assigned to cardiolipin-bound OmpF were observed. No LPS crosslinks were detected in these mutants, consistent with these aromatic girdle residues interacting more closely with PLs. Here, the peaks assigned to FhuA are more abundant, suggesting that OmpF interactions to FhuA are likely mediated by covalently linked PLs or non-covalent LPS bound to FhuA, as detected in spectra. SDS-PAGE gels of samples stained with Coomassie blue and emerald lipid stain are shown as insets, confirming the absence of LPS crosslinks in all cases.
Tabulated native-MS-derived masses for OmpF BPA variants are presented in Supplementary Table   S4.

Fig. S12. BPA incorporation at the extracellular end of OmpF -strands result in limited
lipid crosslinks. Native-MS data of OmpF BPA mutants E234 BPA , Q277 BPA , and D312 BPA purified after UV activation. All samples were injected in a buffer containing 1% -OG and 200 mM ammonium acetate (pH 7.0). For the Q277 BPA mutant, ligand-free and LPS-bound OmpF were observed in the peak 2 sample. Whilst the other OmpF BPA mutants were detected mainly in their ligand-free form in peak 1 and 2 samples. Charge states corresponding to FepA, FhuA, and LPSbound FhuA were observed in the spectrum for the peak 2 fractions of all mutants Although little to no LPS-bound OmpF is detected in these mutants, the presence of co-purified FepA and FhuA suggest that LPS non-covalently bound to monomeric barrels may be mediating these interactions.

SDS-polyacrylamide gels of samples stained with Coomassie blue for protein and ProQ emerald
green for LPS are shown as insets.    and PLs removed from in between the OMPs (pink), with LPS replaced by PLs in the outer leaflet (orange) and the whole SOI (green). The greater distribution of distances from the AFM data reflects the larger dataset from the surface of E. coli compared to the simulated system. The histogram peak maxima of near-neighbor distances from both data sets are nevertheless similar; ~ 9 nm from AFM data, compared to 9.5 nm from the simulations.